Reproducible detection of antigen-specific T cells and Tregs via standardized and automated activation-induced marker assay workflows.

Activation-induced marker (AIM) assays are a promising tool to track antigen-specific T cells, but methodological heterogeneity between research groups hinders their clinical utility. To evaluate AIM assay reproducibility, we conducted a multi-site study of SARS-CoV-2 and cytomegalovirus AIMs. We found inherent variability in AIM assays and optimized approaches to enhance reproducibility, including a standardized workflow to minimize technical variability and a generalizable Box-Cox transformation-based statistical method to optimize calculation of AIM stimulation responses. We further standardized AIM data analysis through the development of automated flow cytometric gating software and demonstrated its superior reproducibility compared to manual analysis. We also characterized antigen-responsive regulatory T cells (Tregs) as CD134+CD137+ cells among CD4+FOXP3+HELIOS+ cells. The combined methodology results in a high degree of reproducibility within and between research groups, providing a comprehensive foundation from which standardized AIM assays can be implemented across diverse scientific and clinical settings.